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doi: 10.1242/10.1242/dev.00189

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A component of the transcriptional mediator complex inhibits RAS-dependent vulval fate specification in C. elegans

Howard Hughes Medical Institute, and Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA

View larger version (115K): [in a new window]   Fig. 1. sy622 and sy665 animals have multiple phenotypes. Scale bars: 100 µm in A-C; 20 µm in D-H. All animals are 1-day-old young adults. (A) Wild-type hermaphrodite. (B) sy622 hermaphrodite. (C) sy665 hermaphrodite. (D) Wild-type male tail. Arrows indicate wild-type rays. (E) sy622 male tail. (F) sy665 male tail. (G) A sy622 male harboring a rescuing dpy-22::gfp transgene that produces a full-length DPY-22::GFP translational fusion protein. (H) A sy622 male harboring a rescuing dpy-22 2548::gfp transgene, which produces a translational fusion between the first 2548 amino acids of DPY-22 to GFP.

View larger version (21K): [in a new window]   Fig. 2. Structure of injected transgenes, and DPY-22 and TRAP230 proteins. (A) Graphical depiction of the PCR fragment amplified from cosmid F47A4 used for rescue experiments. PCR was used to amplify the region between 21115 and 6044 of F47A4. This region contains the entire predicted dpy-22 open reading frame (18942-7241) and parts of the last two exons from F47A4.3. (B) Structural organization of DPY-22 and TRAP230 proteins. Numbers above the proteins refer to amino acid positions. Hatched area represents a glutamine-rich region. Gray areas represent regions of amino acid identity between DPY-22 and TRAP230, as defined by BLAST, with the percentage identity listed below. The positions of the dpy-22 nonsense mutations used in this study are indicated. (C) Structure of DPY-22::GFP fusion proteins encoded by transgenic constructs. DPY-22::GFP is a fusion of the full-length wild-type DPY-22 to non nuclear-localized GFP. DPY-22 2548::GFP and DPY-22 2141::GFP are fusions between the first 2548 and 2141 amino acids of DPY-22, respectively, to non nuclear-localized GFP. Black boxes indicate putative nuclear localization sequences predicted by PROSITE. Hatched area represents a glutamine-rich region. Gray areas indicate GFP amino acids.

View larger version (139K): [in a new window]   Fig. 3. DPY-22::GFP is expressed in the nuclei of vulval cells, and is mislocalized by the dpy-22(sy665) mutation. Left-hand images are Nomarski, and right-hand images are fluorescence. Scale bars: 20 µm. Arrows indicate some of the vulval cells and arrowheads indicate the anchor cells. (A-F) dpy-22(sy622) hermaphrodites rescued with a dpy-22::gfp transgene that encodes a fusion of full-length wild-type DPY-22 to GFP. (A,B) Two-cell stage at the onset of vulval fate specification. (C,D) Four-cell stage of vulval development. (E,F) Eight-cell stage of vulval development. (G,H) dpy-22(sy622) hermaphrodites rescued with a dpy-22 2548::gfp transgene, which encodes a fusion of the first 2548 amino acids of DPY-22 to GFP. (I,J) pha-1(e2123ts)-rescued animals carrying a dpy-22 2141::gfp transgene, which encodes a fusion of the first 2141 amino acids of DPY-22 to GFP. Wild-type DPY-22 and DPY-22 truncated after amino acid 2548 direct GFP to the nucleus, but not DPY-22 truncated after amino acid 2141.